CN1776414B - Electrochemical-based sensor with a redox polymer and redox enzyme entrapped by a dialysis membrane - Google Patents

Abstract

An electrochemical-based sensor includes an electrode with at least one electrode surface, a film disposed on the electrode surface, and a dialysis membrane disposed on the film. The film includes a redox enzyme and a hydrophilic redox polymer (i.e., a polymer with an attached redox mediator(s)). In addition, the dialysis membrane serves to entrap the redox polymer and redox enzyme in the vicinity of the electrode. Such entrapment is accomplished by employing a redox enzyme and a hydrophilic redox polymer of a sufficiently high molecular weight that they do not pass through the dialysis membrane.

Description

Have the redox polymers held back by dialysis membrane and the electrochemical-based sensor of oxidoreducing enzyme

Technical field

The present invention relates generally to sensor, particularly electrochemical-based sensor.

Background technology

Utilize electrochemical-based sensor to determine that the analyte in the fluid sample causes people's very big interest, described electrochemical-based sensor utilizes redox mediators (mediator) and oxidoreducing enzyme and electrode.Think that such electrochemical-based sensor is particularly suitable for the analyte (for example glucose) in the continuous or semicontinuous supervision body fluid sample (for example blood or tissue space fluid sample).For example, electrochemical-based glucose sensor utilizes redox mediators, oxidoreducing enzyme and working electrode, just can determine the concentration of (promptly measuring) glucose with low relatively voltage (for example being lower than 0.4V vs SCE), so reduce any disturbance reponse on working electrode.Can reference example such as United States Patent (USP) 5089112 and 6284478 about further specifying of electrochemical-based sensor, the full content of these patents is included in for reference here.

In typical electrochemical-based sensor, redox mediators promotes electronics to change between the electrode of oxidoreducing enzyme and electrochemical-based sensor.During this period, driven by the analyte that existed, redox mediators and electrode surface, oxidoreducing enzyme is oxidized and be reduced between the state and circulate.The round-robin net result is that electronics is accepted on electrode surface or is given like this, and the simultaneous oxidation reductase keeps the original state of oxidation and catalytic characteristics basically.

In order to measure the analyte in the aqueous sample (for example humoral sample, such as blood, urine or tissue space fluid).The two water-soluble degree of oxidoreducing enzyme and redox mediators is favourable with regard to enough fast reaction kinetics.Therefore, traditional electrochemical-based sensor can be included in the oxidoreducing enzyme and the redox mediators of solvation in the aqueous water sample.

In order to make electrochemical-based sensor have long-term stability,, require redox mediators around electrode, not leach such as continuous or semi-continuous electrochemical-based glucose sensor.In addition, if redox mediators is the material harmful to people or other objects, it is undesirable that redox mediators leaches in the body of people or other objects, therefore should avoid.

In order to prevent to leach, redox mediators is connected on the water-fast synthetic polymer chain, such as polysiloxane.Yet because their hydrophobicity, such chemical constitution activity is low, so be reduced in the reaction kinetics in the water-bearing media.In addition, covalently bound redox mediators on the hydrophilic polymer main chain is not suitable for securely and effectively being attached directly on the electrode of electrochemical-based sensor with traditional approach.

Therefore, also need electrochemical-based sensor as described below in the art, it utilizes oxidoreducing enzyme and redox mediators, but does not exist near the careless problem that leaches oxidoreducing enzyme and/or the redox mediators electrode of electrochemical-based sensor.In addition, the redox mediators of this electrochemical-based sensor and oxidoreducing enzyme should have enough fast reaction kinetics.

Summary of the invention

Electrochemical-based sensor comprises oxidoreducing enzyme and redox mediators according to embodiments of the present invention, but does not exist oxidoreducing enzyme and/or redox mediators from the careless problem that leaches of the electrode of electrochemical-based sensor on every side.In addition, the redox mediators of these embodiments and oxidoreducing enzyme have enough fast reaction kinetics.

A kind of electrochemical-based sensor according to embodiments of the present invention comprises: electrode, and it has at least one electrode surface; Film, it is arranged on the described electrode surface; And dialysis membrane, it is arranged on the described film.Described film comprises oxidoreducing enzyme and water wettability redox polymers (that is: have the redox mediators that is adsorbed, the polymkeric substance of for example covalently bound redox mediators).In addition, dialysis membrane plays a part redox polymers and oxidoreducing enzyme are trapped in electrode on every side.To such an extent as to holding back like this by utilizing enough high oxidoreducing enzyme and the water wettability redox polymers that does not pass through dialysis membrane of molecular weight to realize.

Since oxidoreducing enzyme and water wettability redox polymers the two all by dialysis membrane be trapped in electrode around, can prevent to leach, and electrochemical-based sensor can be used to continuous or semicontinuous measurement (for example 10 hours or longer) in the longer time section.In addition, having under the situation of liquid, aqueous sample, the water-wet behavior of redox polymers provides enough fast reaction kinetics.

Description of drawings

By with reference to detailed description and accompanying drawing that exemplary is described below, can understand the features and advantages of the present invention better, described exemplary is utilized principle of the present invention.Wherein accompanying drawing is:

Figure 1A is the simplification vertical view of a part of the electrochemical-based sensor of typical embodiments of the present invention;

Figure 1B is that electrochemical-based sensor among Figure 1A is along the simplification sectional view ofline 1B-1B;

Fig. 1 C is that electrochemical-based sensor among Figure 1A is along the simplification sectional view ofline 1C-1C;

Fig. 1 D is that electrochemical-based sensor among Figure 1A is along the simplification sectional view ofline 1D-1D;

Fig. 2 is the sketch of the redox polymers of N-vinyl pyrrolidone (NVP) and vinyl ferrocene (VFc) the copolymerization electrochemical-based sensor that is formed for typical embodiments of the present invention;

Fig. 3 illustrates the sketch that VFc is grafted to the redox polymers of the electrochemical-based sensor that is formed for exemplary of the present invention on the polyglycol (PEG);

Fig. 4 illustrates the sketch that a formaldehyde ferrocene (ferrocene carboxaldehyde) is grafted to the reaction sequence on the polyethyleneimine (PEI);

Fig. 5 is the cyclic voltammogram of the electrode tested in the different solutions of the redox polymers that comprises Fig. 2;

Fig. 6 uses the electrode of the redox polymers that is coated with Fig. 2 at the cyclic voltammogram that does not have to obtain under the dialysis membrane situation;

Fig. 7 is the cyclic voltammogram that applies the electrode acquisition of dialysis membrane with the redox polymers that is coated with Fig. 2 then; And

Fig. 8 A and Fig. 8 B represent the glucose catalytic current value of electrode and the funtcional relationship of time, are coated with redox polymers and glucose oxidase among Fig. 2 on the described electrode, and the two all includes dialysis membrane and does not have two kinds of situations of dialysis membrane.Fig. 8 B emphasizes first 10 minutes that 100 minutes shown in Fig. 8 A are measured.

Embodiment

Be expressly understood the present invention for present specification integral body one is made peace, so provide the definition of wherein using term below.

Term " redox mediators " be meant can be with electrode surface and oxidoreducing enzyme the two reduce (reception electronics) or any chemical part of oxidation (giving electronics).

Term " water wettability " is meant any water or aqueous solution to be had the chemical substance or the subgroup (subgroup) of high-affinity.Therefore, hydrophilic compounds is attracted, is dissolved in water or the aqueous solution easily by water or aqueous solution easily, is perhaps absorbed by water or aqueous solution.

Term " hydrophobicity " is meant any chemical substance or the subgroup little to the affinity of water or aqueous solution.Therefore, hydrophobic compound often repels water or aqueous solution, can not be absorbed by water or aqueous solution.

Term " redox polymers " is meant the polymkeric substance of modification (deriving), so that comprise at least a redox mediators.

Shown in Figure 1A to Fig. 1 D is the part of the electrochemical-basedsensor 100 of illustrative embodiment of the present invention.Electrochemical-basedsensor 100 comprises:matrix 102;Reference electrode 104a, it has electrode surface 106a;Working electrode 104b, it has electrode surface 106b; Be arranged onfilm 108 and thedialysis mould 110 that is arranged on thefilm 108 on the electrode surface 106a.Electrochemical-basedsensor 100 also comprisesinsulation course 112 and reference China ink layer (reference ink layer) 114, those skilled in the art can understand: shown in Figure 1A to Fig. 1 D is the part of whole electrochemical-based sensor, too complicated for fear of Figure 1A to Fig. 1 D, the miscellaneous part (for example: shell, analysis/microprocessor assembly and electronic communication circuit) of electrochemical-based sensor is not shown.

Those skilled in the art also can understand with reference to the electrochemical activity layer of Chinaink layer 114 formation withreference electrode 104a one, with respect to the measurement current potential that is applied on the workingelectrode 104b zero potential are set.Have two kinds of electrode forms though those skilled in the art can further understand the electrochemical-based sensor shown in Figure 1A to Fig. 1 D, other electrochemical-based sensor forms known in the art can be used for embodiment of the present invention.

Matrix 102 can be formed by for example polyethylene terephthalate, polybutylene terephthalate thin slice (for example can buy from U.S. GE Plastic), perhaps by oriented polystyrene thin slice (for example can buy from German NSWGmBH company).

Can paste (for example can buying) or any suitable electrochemical reference material by for example Ag/AgCl with reference to Chinaink layer 114 from the Gwent Electronic Materials of Britain Pontypool Wales, comprise that (for example: silver, copper, titanium and lithium) material still is not limited to this to the metal that contains formation part soluble-salt.

Butinsulation course 112 can be stuck with paste (for example can buy from Sericol InksLtd.) by the China ink of for example serigraphy ofinsulation.Reference electrode 104a and workingelectrode 104b can be with well known to a person skilled in the art that any suitable material makes, and forexample reference electrode 104a and workingelectrode 104b can be made such as the carbonaceous conductive China ink by conductive ink.

Reference electrode 104a, workingelectrode 104b,insulation course 112 anddialysis mould 110 can have any suitable thickness.But, in each the scope of typical thickness between 1 micron to 100 microns of these layers.

Film 108 comprises oxidoreducing enzyme and water wettability redox polymers (not shown in figure 1).Utilize dialysis mould 110 andinsulation course 112 that the redox polymers offilm 108 and oxidoreducing enzyme are trapped near the workingelectrode 104b.

To such an extent as to the water wettability redox polymers offilm 108 and the molecular weight of oxidoreducing enzyme enough high they substantially can not be by the mould 110 (hereinafter further specifying) of dialysing.Water wettability redox polymers and oxidoreducing enzyme can have any suitable molecular weight, as long as the given dialysis membrane of water wettability redox polymers is held back.Usually, the molecular weight of described water wettability redox polymers is greater than about 10kg/mol (10,000 dalton), and the molecular weight of described oxidoreducing enzyme is preferably greater than 80,000 dalton (80kg/mol) greater than 5000 dalton (5kg/mol).About this, notice that the molecular weight of glucose oxidase (a kind of oxidoreducing enzyme) is approximately 160,000 dalton (160kg/mol).

Adjustdialysis membrane 110, make and to have only the low relatively compound of molecular weight (for example glucose) to pass through, simultaneously the high relatively compound (for example the water wettability redox polymers and the redox mediators of film 108) of molecular weight be retained in workingelectrode 104b near.Dialysis membrane also can play analyte (for example glucose) diffusion-controlled layer and ELIMINATION OF ITS INTERFERENCE layer.

Dialysis mould 110 can be any suitable dialysis mould well known in the prior art, comprises cast polymerization thing dialysis membrane or cross-linked polymer dialysis membrane.The non-limitative example of the dialysis membrane that is fit to comprises: (i) casting dialysis membrane, and it is formed by the acetone soln that contains 2% (w/v) cellulose acetate (CA) and 0.7% (w/v) polyglycol (PEG); (ii) cross-linked polymer dialysis membrane, its 2-aqueous isopropanol by the polyethyleneimine that contains 5% (w/v) (PEI) and poly-(propylene glycol) diglycidyl ether (PPGDGE) of 0.7% (w/v) forms.

The water wettability redox polymers that is applicable tofilm 108 can be by for example forming to the high relatively polymkeric substance of molecular weight redox mediators is covalently bound.The water-wet behavior of described water wettability redox polymers is convenient to promote the redox mediators of water wettability redox polymers and the favourable reaction between the oxidoreducing enzyme, so that obtain enough fast reaction kinetics.

The oxidoreducing enzyme offilm 108 can be any suitable oxidoreducing enzyme that those skilled in the art understand.For example, non-limiting instance comprises: glucose oxidase, lactic acid (latate) oxidase, bilirubin oxidase, sarcosine oxidase, choline oxidase, cholesterol oxidase and xanthine oxidase, and glucose dehydrogenase, alcohol dehydrogenase, peroxidase (for example horseradish peroxidase) and catalase.

The benefit of the electrochemical-based sensor of embodiment of the present invention is preparation and processing easily of the component in the solution in manufacture process.Therefore, for example can make described electrochemical-based sensor with traditional printing and paint-on technique.

Fig. 2 is the synoptic diagram that the process that forms theredox polymers 200 that is applicable to the electrochemical-based sensor in the exemplary of the present invention is shown.As shown in Figure 2, can pass through with 2 N-vinyl pyrrolidone (NVP) hydrophilic monomer that 2 '-azoisobutyronitrile (AIBN) causes and the free-radical polymerizedformation redox polymers 200 of vinyl ferrocene (VFc) redox mediators.Other hydrophilic monomers that those skilled in the art understand, for example, acrylamide monomer, hydroxyethyl methylacrylate monomer and polyglycol (PEG) macromonomer can replace NVP.

The mol ratio of NVP in Fig. 2: VFc is expressed as m: n, for example can be from about 100: 1 in about 100: 5 scope.Should be noted that if the ratio of VFc greater than about 5%, the redox polymers of generation may become and is not dissolved in some aqueous sample.In addition, if the ratio of VFc less than about 1%, the redox conductivity ofredox polymers 200 can become too low, so that can not guarantee to measure the electron exchange speed that glucose needs.The water-wet behavior that shall also be noted that NVP givesredox polymers 200 high relatively water wettability.

The redox polymers that is applicable to the electrochemical-based sensor of embodiment of the present invention can comprise any suitable redox mediators, comprise: osmium complex, quinone, ferricyanide, methylenum careuleum, 2,6-dichloroindophenol, thionine, gallocyanin, indophenols and their combination, but be not limited thereto.In addition, this redox polymers can be formed by any suitable hydrophilic monomer, for example includes but not limited to have the hydrophilic monomer of acrylate or vinyl polymerizable functional group.The example of other hydrophilic monomers that are suitable for comprises: hydroxyethyl methylacrylate, N-N-isopropylacrylamide, glyceral methacrylate and acrylamide.For different amboceptors and hydrophilic monomer, the process shown in Fig. 2 should correspondingly change.

For example, can also be by (for example polyglycol (PEG), polyvinylpyrrolidone or polyethyleneimine (PEI) are grafted to the redox polymers that forms the electrochemical-based sensor that is applicable to embodiment of the present invention on the redox mediators hydrophilic polymer.Fig. 3 illustrates VFc is grafted to the reaction sequence that PEG goes upformation redox polymers 300, and describedredox polymers 300 is applicable to the electrochemical-based sensor of exemplary of the present invention.In the reaction sequence shown in Figure 3,, formredox polymers 300 to capture mechanism by hydrogen having under the cinnamic prerequisite with benzoyl peroxide as initiating agent.

Shown in Fig. 4 is that PEI is grafted to the reaction sequence that formsredox polymers 400 on the formaldehyde ferrocene, and describedredox polymers 400 is applicable to the electrochemical-based sensor of embodiment of the present invention.In the reaction sequence shown in Figure 4, the aldehyde group of the secondary amine group of PEI and formaldehyde ferrocene forms schiff bases (being imines).Because the schiff bases key forms instability, receive (NaBH with the hydrogen boronation₄) imines is reduced into tertiary amine.

The following examples further specify and prove the others and the benefit of the electrochemical-based sensor of embodiments of the invention.

Embodiment 1

In-0.1 to 0.5V scope, carry out cyclic voltammetry (CV) with 5% solution (in phosphate buffer (PBS)) of the redox polymers among Fig. 2 200 and glassy carbon electrode (GCE) at E/Vvs.Ag/AgCl with 50mV/s speed.Thereby obtain thecurve 500 of Fig. 5, and confirm thatredox polymers 200 has redox active on the glassy carbon electrode.

Then, glucose oxidase (a kind of oxidoreducing enzyme) is added among the PBS to concentration reach 0.05wt%.Shown in thecurve 510 of Fig. 5, the redox peak value of CA scanning afterwards reduces slightly.The oxidation ofcurve 510 is compared with the redox peak value ofcurve 500 slightly with the reduction peak value and is reduced, and can think owing to add the result that glucose oxidase dilutes a little and/or because the absorption of glucose oxidase causes fraction GEC passivation.

Then, glucose is added among the PBS to concentration reach 100mM.Shown in theCV curve 520 of Fig. 5, add glucose and cause that oxidation current improves.Because glucose oxidase partly is converted to the ferrocene part to all ferricenium on the electrode surface, can't see reduction wave on curve 520.The character shape ofcurve 520 also can be called catalytic wave, and maximum current is directly proportional with concentration of glucose in this waveform.

Embodiment 2

GCE is immersed in the solution of theredox polymers 200 that contains 5% Fig. 2, describedredox polymers 200 is dissolved in the 2-isopropyl alcohol.Then, from 5% solution, take out GCE, and make its drying.Then, GEC is immersed among the PBS, in-0.1 to 0.5V scope, utilize the test of CV method with 20mV/s speed at E/V vs.Ag/AgCl.As shown in Figure 6, the redox peak value reduces rapidly in CV scans continuously.This shows, is not having the dialysis membrane adsorption film to hold back under the situation ofredox polymers 200 around GCE, andredox polymers 200 has been washed GCE off.

Embodiment 3

By 5% (w/v) solution that 0.5 μ L is dissolved in theredox polymers 200 in the 2-isopropyl alcohol be coated in carbon electrode (on the 2.25mm * 2.25mm), then indrying box 50 ℃ dry about 5 minutes down, prepare electrode coated.Then, the acetone soln that contains 2% (w/v) cellulose acetate (CA) and 0.7%PEG by preparation forms dialysis membrane on electrode coated.

Then, 0.8 μ L CA/PEG potpourri be coated onto this electrode coated on, and indrying box 50 ℃ dry 30 minutes down.The CA/PEG potpourri forms the casting dialysis membrane, described casting dialysis membrane theredox polymers 200 of macromolecule be retained in carbon electrode around.The GCE of above-mentioned preparation is immersed among the PBS immediately, in-0.1 to 0.5V scope, utilize the CV method to survey with 20mV/s speed at E/V vs.Ag/AgClExamination.As shown in Figure 7, beginning to descend gradually then in the CV scanning continuously because electrode incipient wetness redox peak value raises.Should be noted that rate of descent observed in Fig. 7 is lower than viewed rate of descent among Fig. 6.This shows that dialysis membrane among this embodiment helpsredox polymers 200 is trapped near the carbon electrode.

Embodiment 4

5% (w/v) solution of theredox polymers 200 of 0.5 μ L be coated onto carbon electrode (on the 2.25mm * 2.25mm), then indrying box 50 ℃ dry about 5 minutes down, preparation does not have the electrochemical-based glucose sensor of dialysis membrane.Then, the solution of glucose oxidase in PBS of 1μ L 10% (w/v) is coated onto on the electrode, following dry 10 minutes at 50 ℃ in drying box.

Embodiment 5

Prepare electrochemical-based glucose sensor with the method that is similar among theembodiment 4, just on this electrochemical-based glucose sensor, be coated with dialysis membrane.More specifically, above coated dialysis membrane be cast polymerization thing film, form by the formulations prepared from solutions that in acetone, contains 2% (w/v) CA and 0.7%PEG.Described dialysis membrane is to coat the described CA/PEG potpourri of 0.8 μ L on example 4 described electrochemical-based glucose sensors, makes in 30 minutes at 50 ℃ of following dry described sensors in drying box then.CA and PEG solution form the casting dialysis membrane, andredox polymers 200 and oxidoreducing enzyme (being glucose oxidase) that it keeps macromolecule allow the analyte (such as glucose) of small-molecular weight to pass through simultaneously.

Embodiment 6

Electrochemical-based glucose sensor (dialysis membrane is arranged) at electrochemical-based glucose sensor (not having dialysis membrane) that hasindependent detection embodiment 4 under the 100mM glucose condition and embodiment 5.At E/Vvs.Ag/AgCl working electrode with each electrochemical-based sensor of 20mV/s rate scanning in-0.1 to 0.5V scope, be exposed in the air, at room temperature simultaneously.The catalysis waveform of every interval record generation in 10 minutes in 100 minutes.

Fig. 8 A and Fig. 8 B illustrate the oxidation current that the catalysis waveform shows the electrochemical-based glucose sensor that does not have dialysis membrane to be reduced fast.This means thatredox polymers 200 is washed off from the electrode of so electrochemical-based glucose sensor.Yet, for the electrochemical-based glucose sensor that comprises dialysis membrane, the catalysis waveform show oxidation current descend slower, thereby show that redox catalysis amboceptor and oxidoreducing enzyme are retained in around the electrode by dialysis membrane.

Embodiment 7

Have the electrochemical-based glucose sensor of dialysis membrane with the preparation of the method forsimilar embodiment 5, be described dialysis membrane be the crosslinking polymer thin film that comprises PEI and PPGDGE.In order to form such dialysis membrane, the PPGDGE of the PEI of 52mg and 106mg is mixed, so that in the 2-of 1mL isopropyl alcohol, form the potpourri of PEI/PPGDGE, then, the PEI/PPGDGE potpourri of 0.8 μ L is coated onto on the carbon coating electrode (as what prepare in the foregoing description 4), and following dry 30 minutes at 50 ℃ in drying box.PEI and PPGDGE solution form dialysis membrane with the form of cross-linked polymer dialysis membrane, and this dialysis membrane keeps theredox polymers 200 and the oxidoreducing enzyme of macromolecule, allows the analyte (such as glucose) of small-molecular weight to pass simultaneously.

Imagination causes swelling when film contacts aqueous solution by the water-wet behavior of the cross-linked polymer dialysis membrane that PEI and PPGDGE solution form.This swelling makes the cross-linked polymer dialysis membrane have the feature of hydrogel.Therefore, the film of generation can be called hydrogel layer.In addition, it is faster that the analyte that molecular weight is low relatively (for example glucose) penetrates the speed of described hydrogel layer redox polymers and the oxidoreducing enzyme higher relatively than molecular weight.

Embodiment 8

2 of the vinyl ferrocene (VFc) of NVP, the 0.87g of the redox polymers that is applicable to electrochemical-based glucose sensor of the present invention by using 10.4g and 0.11g, the free radicals copolymerization reaction of 2 '-azoisobutyronitrile (AIBN) synthetic (see shown in Fig. 2 in proper order).Described being reflected in the round-bottomed flask carried out.Before reaction causes, in reaction solution, fed nitrogen bubble one hour, remove oxygen wherein.In nitrogen atmosphere, reaction flask is heated to 70 ℃ in oil bath then and reaches 24 hours, simultaneously successively geomagnetic disturbance.

The redox polymers that produces is dissolved in the methylene chloride, and from solution, is settled out with ether.Then, the redox polymers of precipitation is filtered, dry down at 50 ℃ in drying box.By the deionized water dialysis small-molecular weight in the redox polymers is partly removed then.Dialysis tube be by molecular weight be the tunica fibrosa of 16Kg/mol.

Embodiment 9

By the synthetic redox polymers (as shown in Figure 3) that is applicable to the electrochemical-based sensor of the embodiment of the invention of graft reaction.At first, the VFc of 0.6g is dissolved in preparation VFc solution in the 2.1g styrene.In another container, suspension 0.24g benzoyl peroxide (70%) preparation benzoyl peroxide suspending liquid in 1.37g dimethylbenzene.In another container, the PEG of 10g (molecular weight is 8Kg/mol) and 20g ethoxy ethanol are mixed, be heated to 90 ℃ then, preparation PEG solution.

Then, in the PEG solution of 90 ℃ of VFc solution addings, form the VFc/PEG potpourri.After adding VFc solution, benzoyl peroxide suspending liquid is joined in the VFc/PEG potpourri to form reaction mixture at once.In case initiation grafting, reaction mixture forms single-phase, and color is a salmon pink.90 ℃ of followingstirring reaction mixtures 2 hours, stir a night continuously at 70 ℃ then.Then, reaction mixture is converted into the analysis pure water of 50ml, forms the red/brown emulsion that contains redox polymers.During the electrochemical-based glucose sensor of preparation one embodiment of the present of invention, red/brown emulsion is applicable to and applies the GCE film.

Embodiment 10

By the synthetic another kind of redox polymers (as shown in Figure 4) that is applicable to the electrochemical-based sensor of embodiments of the invention of graft reaction.In described building-up process, 0.3g formaldehyde ferrocene is mixed with the PEI of 50ml absolute methanol and 18g, form reaction solution.The number-average molecular weight of PEI (Mn) is 10Kg/mol, and weight-average molecular weight (Mw) is 25Kg/mol.

Then, stirring reaction solution is 2 hours in air, also carries out thin-layer chromatographic analysis (TLC) with the methyl alcohol eluate by the aliquot of removing reaction solution and monitors reaction.The TLC assay shows formaldehyde ferrocene reaction becoming imines all after 2 hours.

Then, the 100mg borohydride sodium is added in the 10ml methyl alcohol, will in its adding reaction solution imines be reduced into tertiary amine then.After adding borohydride sodium, again reaction solution was stirred 2 hours, add 15ml water then gradually.Then, in order to guarantee whole borohydride sodium reactions, add 20ml water more rapidly.Then with 100ml diethyl ether extractive reaction solution to remove any possible organic impurities, abandon organic phase then.After extraction step, reaction solution is applicable to the redox polymers of the electrochemical-based sensor of embodiment of the present invention a dry night with production in 70 ℃ drying box.

The various alternative that should be appreciated that embodiment of the present invention described herein can be used to implement the present invention.Being intended that of claim of the present invention limits scope of the present invention, so contains interior structure of these claim scopes and their equivalent structure.

Claims (4)

1. electrochemical-based sensor comprises:

Electrode, it has at least one electrode surface;

Film, it is arranged on the described electrode surface, and described film comprises:

Oxidoreducing enzyme; With

The water wettability redox polymers; And

Dialysis membrane, it is arranged on the described film,

Wherein, described dialysis membrane is used for described oxidoreducing enzyme and water wettability redox polymers are trapped in around the electrode,

The molecular weight of wherein said oxidoreducing enzyme and water wettability redox polymers makes described oxidoreducing enzyme and water wettability redox polymers can not pass through described dialysis membrane, thus described oxidoreducing enzyme and water wettability redox polymers are trapped in around the electrode

Wherein said redox polymers obtains by N-vinyl pyrrolidone and vinyl ferrocene copolymerization.

2. the electrochemical-based sensor of claim 1, it further comprises working electrode.

3. electrochemical-based sensor comprises:

Electrode, it has at least one electrode surface;

Film, it is arranged on the described electrode surface, and described film comprises:

Oxidoreducing enzyme; With

The water wettability redox polymers; And

Dialysis membrane, it is arranged on the described film,

Wherein, described dialysis membrane is used for described oxidoreducing enzyme and water wettability redox polymers are trapped in around the electrode,

The molecular weight of wherein said oxidoreducing enzyme and water wettability redox polymers makes described oxidoreducing enzyme and water wettability redox polymers can not pass through described dialysis membrane, thus described oxidoreducing enzyme and water wettability redox polymers are trapped in around the electrode

Wherein said redox polymers is by being grafted to vinyl ferrocene synthetic obtaining on the polyglycol.

4. electrochemical-based sensor comprises:

Electrode, it has at least one electrode surface;

Film, it is arranged on the described electrode surface, and described film comprises:

Oxidoreducing enzyme; With

The water wettability redox polymers; And

Dialysis membrane, it is arranged on the described film,

Wherein, described dialysis membrane is used for described oxidoreducing enzyme and water wettability redox polymers are trapped in around the electrode,

The molecular weight of wherein said oxidoreducing enzyme and water wettability redox polymers makes described oxidoreducing enzyme and water wettability redox polymers can not pass through described dialysis membrane, thus described oxidoreducing enzyme and water wettability redox polymers are trapped in around the electrode

Wherein said redox polymers is gone up synthetic obtaining by the formaldehyde ferrocene being grafted to polyethyleneimine (PEI).

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